Nutrient availability affects cell growth, proliferation and survival. Apart from pancreatic beta cells, skeletal myocytes, adipocytes and hepatocytes, there is a dearth of information about how cells respond to positive energy balance in the whole animal. Using endothelial cells as a prototypic cell lineage that is resistant to transformation, we will characterize cellular responses to the availability of a nutrient source, glucose, in excess of metabolic requirements (relevant to Projects 2, 3). High concentrations of external glucose trigger an autoregulatory mechanism to limit both glucose uptake and growth factor signaling, while stimulating intracellular pathways that regulate cell proliferation and apoptosis (examined in Project 2). Specifically, we have found that incubation of primary endothelial cells in 25 mM glucose induces c-myc transcription, activation of NF-kB pro-inflammatory pathways and downregulation of PI3-K/Akt signaling (relevant to Projects 2,4) in this cell type. These findings extend a growing appreciation that oncogenes, tumor suppressor genes, and signal transduction pathways are tightly coupled to changes in nutrient availability and energy metabolism. Having established how proliferative responses are coupled to nutrient excess in cells that resist transformation, we will investigate how primary mammary epithelial cells respond to excess glucose availability/We hypothesize that cells that are susceptible to neoplastic transformation will exhibit distinct identifiable responses to nutrient excess that favor cell proliferation. Finally, we will determine whether immortalized, initiated mammary epithelial cells exhibit heightened sensitivity to the effects of glucose excess, providing an epigenetic mechanism for tumor promotion. This project addresses key questions for obesity and cancer risk: how nutrient excess is coupled to changes in cell proliferation and survival, and at which point in carcinogenic progression does energy balance become critical. Insights gained from these investigations will provide novel frameworks for analyzing the effects of obesity on site-specific cancers in animal models and human populations (relevant to Projects 2-5).